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Identification of Bacteria from Oral and Rectal Swabs from Different Species of Rodents in Kemasul Forest Reserve, Pahang

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Identification of Bacteria from Oral and Rectal Swabs from Different Species of Rodents in Kemasul Forest Reserve, Pahang Journal of Wildlife and Parks, 33: 75-93 (2018) 75 IDENTIFICATION OF BACTERIA FROM ORAL AND RECTAL SWABS FROM DIFFERENT SPECIES OF RODENTS IN KEMASUL FOREST RESERVE, PAHANG *Farah Shafawati Mohd-Taib1, Rosha Asyikha Mohd Sham1, Hasnorsharmini Hassan1 & Wan Syaidatul Aqma2 1School of Environmental Science and Natural Resources, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia. 2School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia. *Corresponding author’s email: [email protected] ABSTRACT Small mammals such as rodents have potential in carrying disease caused by bacteria and this needs to be addressed to prevent serious disease outbreaks. Forest fragmentation by human activities adversely affected the flora and fauna diversity which indirectly increasing disease transmission to the living things in the forest and its surrounding. This research aims to determine the presence of bacteria in oral and rectal swabs of selected rodent species and to investigate the antibiotic susceptibility of the isolated bacteria. This study was conducted in Kemasul Forest Reserve, Pahang and trappings were conducted from August to November 2015. Swab samples were taken and bacterial identification was made in the laboratory by using biochemical test and Analytical Profile Index (API) kit. Staphylococcus aureus, Staphylococcus epidermidis, Enterobacter cloacae, Pseudomonas luteola, Corynebacterium kutscheri and Bacillus spp. were identified, and there were no different bacteria species in the two different anatomical sites. These bacteria are normal commensal bacteria and opportunistic pathogens. Antibiotic susceptibility test indicated that ciprofloxacin, and vancomycin are the most susceptible antibiotic for all bacteria. This knowledge will enhance understanding of bacterial infection in rodents and becomes important baseline data for bacteria species. Keywords: Bacterial identification, antibiotic susceptibility, disease transmission, oral and rectal swabs of rodents 76 Farah Shafawati Mohd-Taib, Rosha Asyikha Mohd Sham, Hasnorsharmini Hassan & Wan Syaidatul Aqma Received (14-February-2017); Accepted (02-August-2017); Available online (08-June-18) Citation: Mohd-Taib, F.S., Mohd Sham, R.A., Hassan, H. & Aqma, W.S. (2018). Identification of bacteria from oral and rectal swabs from different species of rodents in Kemasul Forest Reserve, Pahang. Journal of Wildlife and Parks, 33: 75-93. INTRODUCTION Rodents are a group of mammals that are successfully exploiting diverse habitat and environment for their survival worldwide. Most rodent species live in the wild with less direct interaction with human being. There are some species found in the wild including Maxomys rajah, Maxomys surifer, Leopoldamys sabanus, Sundamys muelleri and Hylomys suillus (Mariana et al., 2008; Zain et al., 2015). Meanwhile, some rodents’ species have adapted to live in urban areas close to human settlement such as Rattus rattus, Rattus norvegicus, Maxomys whiteheadi, Mus musculus and Mus domesticus. Sahimin et al. (2014) reported that Rattus rattus diardii is the dominant domestic rats in urban areas in Malaysia. Previous studies by Castillo et al. (2003) reported that 74% of commensal rodents were captured in the urban areas. M. domesticus was the most abundant species followed by Calomys musculinus which was related to open areas such as railway bank, streams bank and rubbish dump. Furthermore, they are considered as pests that eat crops, animal droppings, garbage, and other rats. Additionally, rodents are well known as carriers of a variety of diseases that can infect both humans and livestock. Many of these rodent species are also reservoirs that cause debilitating diseases in human and livestock (Singleton et al., 2003). These rodents can spread and transmit disease as they live close to human habitat. Consequently, rodents can vector more than 60 known diseases (Phan et al., 2011), some of which are zoonotic diseases, caused by bacteria that pose a risk to human health and could lead to death. For instance, Aeromonas hydrophila bacteria can be transmitted from rodents to human which can cause gastroenteritis with diarrhoea symptoms (McCoy et al., 2010) and Leptospira spp. can cause death due to renal failure, cardio pulmonary failure and haemorrhage (Holt et al., 2006). Kemasul Forest Reserve, located in Pahang, has been converted to monoculture plantation and leaving some forest remnants scattered within the forest. Opening areas for this project resulted in the separation of the forest into small fragments that caused disturbance to wildlife that lives in the forest fragments and forced Journal of Wildlife and Parks, 33: 75-93 (2018) 77 them to approach human settlements near the forest and resulting conflict between human and animal. These animals have undergone long series of physiological stress due to habitat disturbance and therefore easily infected with diseases. Furthermore, as human activities within the forest increase, there has been a growing concern if the natural environment has been adversely affected. Habitat disturbance may increase bacterial infection to the animals due to their poor health condition. As rodent are hosts and potentially be infested with bacteria, there are high possibilities that disease may present among these species and may cause deleterious effects to other living things surround them. Hence, the objectives of this study are to isolate and identify bacteria of different anatomical sites of rodents and to investigate the antibiotic resistance of the isolated bacteria. METHODOLOGY Study Areas Kemasul Forest Reserve in Pahang (Figure 1) was selected as a case study. The study was conducted in two different locations which were Chemomoi and Jambu Rias. Latitude and longitude for Chemomoi are 03°19ʹ34.2ʺN, 102°15ʹ0.16ʺE and for Jambu Rias are 03°27ʹ18.3ʺN, 102°08ʹ175ʺE (Figure 2). Kemasul Forest Reserve has a total area of 39,000 hectares includes areas of Temerloh, Bera, and Bentong (Figure 2). The whole area was gazetted as Forest Farm project under the supervision of the Pahang Forestry Department. Forest Project was privatised in 1994. Figure 1 Location of Kemasul FR in Pahang State in Peninsular Malaysia. 78 Farah Shafawati Mohd-Taib, Rosha Asyikha Mohd Sham, Hasnorsharmini Hassan & Wan Syaidatul Aqma Figure 2 Jambu Rias (JR) and Chemomoi (CM) in Kemasul Forest Reserve, Pahang Sampling Procedure in the Field Trapping was employed from August to November 2015. Rodents were captured using small wire mesh cage traps. A total of 300 traps were deployed at the study sites for two weeks continuously for three months. Traps were placed above ground level, including on tree stumps, tree branches or fallen log. Baits used were oil palm fruits and jackfruits. The captured rodents were anaesthetised with about 0.5-1 mL of Zoletil (active ingredients; tiletamine and zolazepam). Then, weight and morphometric measurements of head and body, tail, ear, hindfoot and physical appearances were recorded. Each animal’s age, sex, and species were identified following a guide authored by Francis (2008). Swab samples were obtained from the oral and rectal mucosa of each rodent using sterilised swabs for collection of bacteria. UKM Animal Ethics Committee approved all procedures with approval number FST/2016/SHUKOR/18-MAY/750-MAY-2016-SEPT.- 2018-AR-CATS. All captured individuals were released at the point of capture after they gained consciousness. Only four rodents species were selected in this study namely M. rajah, M. surifer, M. whiteheadi and R. rattus. The first two species were forest species, while the later were commensal species near human settlements. Journal of Wildlife and Parks, 33: 75-93 (2018) 79 Bacterial Identification Serial dilution method was used where a series of sequential dilutions used to reduce a dense bacteria solution. Swab sample of different species was dipped in 99 mL of sterilised saline solution and diluted till 10-6 dilution. A 1 mL pipette was used to transfer the sterilised saline contained culture into the 10-1 bottle. The bacterial suspension was mixed thoroughly using the vortex mixer. The volume of 0.1 mL of the final three dilutions (10-2, 10-4, and 10-6) was transferred into the nutrient agar plates and was spread evenly over the entire surface of the nutrient agar plates until the medium no longer appears moist. The triplicate plates were incubated at 37˚C for 24 hours. Next, streak plate method was used to obtain single colonies of bacteria for isolation of pure bacteria cultures (Sanders, 2012). The sample was picked and spread over about one-quarter of the surface of the medium and repeated until all four quarters were covered. The plate was labelled and incubated inversely at 37˚C for 24 hours. The colony forming units (CFU) for each plate were counted manually after incubation period was completed prior to observation. One of the basic steps in identifying bacteria is observing its colony morphology such as colour, size, form, elevation, and margin. According to Chauhan (2012), Gram staining is an empirical method of differentiating bacterial species into two large groups of Gram-positive and Gram-negative bacteria. One single colony was placed on a clean glass slide. The smear was air-dried and heat fixed. The slide was treated with crystal violet for 1 minute followed by washing with running water. Then, iodine solution was used to cover the slide for 1 minute
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